It is well-known, that biological materials in solutions such as vaccines are susceptible to varying influences including heat, oxidizing reagents, salts, pH, light, and proteolitic enzymes.
Several methods are known for reducing these detrimental effects in general and more specifically to improve the stability of a vaccine especially during storage. For example, storage below 0° C., and as low as −70° C., in a freezer is a well-known method. At even lower temperatures, e.g. in liquid nitrogen, many biological materials including living cells can successfully be stored for many years. However, such methods are not always convenient in those situations, for example, that involve the innoculation of free-ranging livestock.
Lyophilizing or freeze drying is another known way of conserving live cells and viruses for use as vaccines. During freeze drying, the solution containing the biological material is first frozen and the water is then evaporated by sublimation, usually under high vacuum and sub-zero temperatures. Previous approaches have used freeze drying or other techniques to formulate viral vaccines but still pose other difficulties with respect to preparation and administration of a stable final dosage form.
For example, U.S. Pat. No. 4,251,509 (Hanson and Abegunde) discloses a stable particulate viral vaccine intended to be orally administered to free ranging livestock in a dry state. However, the disclosed dosage form is not freeze dried but prepared by concentrating and extruding a paste dried to form pellets.
Antioxidants, the selection of which will depend on the particular virus, are required to promote thermostability. Such a formulation may be more complicated to prepare and is not particularly well-suited for those instances when it is desired to dissolve the vaccine before immunization.
U.S. Pat. Nos. 3,458,621 and 3,608,030 (Tint) disclose the use of freeze dried virus preparations to prepare a tablet for oral administration for the immunization of the intestinal tract. However, the tablets prepared are for oral administration and are provided with an enteric coating to delay disintegration. During in-vitro testing they did not rapidly disintegrate and were found to disintegrate only “within 25 minutes” with simulated intestinal fluid. Moreover, the time to disintegrate was measured in simulated intestinal fluid thereby discounting effects of the enteric coating. In addition, Tint further emphasizes that unless the tablets are “press coated” they will lose their titer as shown by a comparison between press coated and non-press coated tablets. Tint notes that the non-press coated tablets “not only failed to elicit an antibody response in all the antibody-negative individuals, but, in addition, the magnitude of the titer rise was significantly smaller.” (See column 4, line 40).
PCT Publication No. WO 99/21579 (Seager, et al.) assigned to the R.P. Scherer Corp. discloses a “fast” dispersing composition for a veterinary vaccine such as against New Castle disease that is freeze dried and “loosely compacted.”
The dosage form is disclosed as an “open matrix network”, such as a “solid toam” referenced from U.S. Pat. No. 4,371,516 (counterpart to UK Patent No. 1,548,022), as opposed to a compressed form or hard tableting. Moreover, the vaccines are directed to oral administration and targeted towards retention at mucosal tissue. Adjuvants serve to provide sufficient residence time for absorption thereon. The disclosed vaccine formulation does not provide for preparation of a liquid dosage for later immunizing, nor a means to readily form a stable, measured vaccine solution for administration as a liquid dose, nor for providing a stable compressed or hard tableted lyophilisate to facilitate later administration thereof.
U.S. Pat. No. 5,587,180 (Allen, Jr. et al)describes a process for making a particulate support matrix for a rapidly dissolving tablet. The process teaches away from freeze drying and uses standard spray-drying techniques. The particulate support matrix is suitable for dosage administration when placed into the oral cavity. Moreover, no stable vaccine formulation is provided as a vaccine solution in a liquid dosage form.
U.S. Pat. No. 5,336,666 (Neway et al.) discloses a freeze dried liquid vaccine that may form a tablet to be reconstituted in liquid form. However, the vaccine is limited to a polar glycopeptide of a particular bacterium and does not provide for complete or rapid dissolution.
Although freeze drying of biological material can be performed according to lyophilizing procedures well known to one skilled in the art to provide a stable vaccine preparation, the titer of a live virus at the end of lyophilization is typically not the same as it was for the solution before the lyophilization process. In general it is not possible to conduct titration before lyophilization as the solution is not stable until it is freeze dried. In addition, the titer will change unpredictably during lyophilization. Consequently, an estimate for the initial titer based on experience is only validated by titration after freeze drying. As a result of all of the above, it is almost impossible to achieve a defined accurate target titer.
If lyophilization has been carried out with the lyophilisate already contained in vials, reworking of the batch is typically not possible, and in some cases a whole batch must be discarded if it is not up to specification.
U.S. Pat. No. 5,897,852 (Wilderbeek, et al.) attempts to solve such a problem using different “freeze dried bodies” with “lyospheres” to make up for shortfalls in the titer of a lyophilized cake. However, each lyosphere has its own titer thereby necessitating the titration of multiple bodies to arrive at a desired titer. Even in the best case, this method does not achieve the exact required titer as it is only an approximation due to the use of combined bodies to achieve the target titer. Furthermore, the production of lyospheres is relatively difficult compared with the more straightforward freeze drying of solutions to produce a cake or powder. Additionally, a special matrix is often required to prevent the lyosphere material from being pulverized after drying. In general, the method requires the preparation of separate solutions each having a different titer, additives and adjuvants. The method does not solve the problem that the titer for a batch of live virus is rarely homogenous, and varies from one vial to the next, particularly over the areas of the cold plates in the lyophiliser. Therefore, determination of the number of lyospheres needed per vial is always an approximation.
Freeze drying may also be useful for vaccines comprising more than one immunogenic component. For example, EP 290197A discloses a freeze dried tetravalent vaccine. The procedure discloses the mixing and subsequent freeze drying of four live virus vaccine components.
A disadvantage to current freeze drying techniques for vaccine preparation is that the process is very complex, having many variables, rendering it notoriously difficult to perform in a reproducible manner to achieve acceptable product and dosage uniformity. This is especially problematic for veterinary vaccines where a large number of doses are freeze dried in one vial. The problem is less common for human vaccine preparation, although equally appreciable where mass innoculation is necessary, for example in military scenarios, or in situations of pandemic infection. For example, a typical single vaccine vial for poultry vaccination comprises either 1000 or 2000 doses, and is registered as such. Before freeze drying, a rough estimation is made about the titer of the material, but the final titer can only be determined after freeze drying, as the titer often decreases rather unpredictably during freeze drying. Furthermore, the raw material of live virus or bacteria cannot usually be kept stable long enough to obtain accurate titer results before freeze drying.
In practice, a container originally comprising more than 2000 doses can often turn out to comprise only 1900 doses after freeze drying. In that case, the vial might only be marketable as a 1000 dose vial, since that typically is the only other official registrable dosage. For such a scenario, a 47% waste of material and corresponding increase in costs could occur. This problem is compounded with combination vaccines because the dosage of the component with the lowest titer must be used to characterize the entire batch. As a further disadvantage, when vials intended to provide 2000 single doses are subsequently marketed as 1000 dose vials and used to innoculate 1000 animals, the animals thus dosed are unnecessarily over-exposed. Moreover, deliberately increasing the number of doses before freeze drying is not a satisfactory alternative when some of the vaccine batch is dried more efficiently. In that situation, one simply ends up wasting that material.
These problems will become increasingly important to solve as Registration Authorities currently work toward a registration system where the number of vaccine dose titers are set between well-defined upper and lower limits. Given the many variables in both the production and the freeze drying process, it will be difficult to meet these limits on a large-scale production basis. As indicated, this problem will be even more pronounced when a combination vaccine is required.
Yet a further disadvantage to previous freeze dried combination vaccines is that a large number of formulated combinations must be kept in storage. This occurs because the various components in a combination vaccine are typically mixed prior to freeze drying. Thus, for the preparation of a full range of single/multi-component-vaccines against, e.g. two diseases, three different products must be kept in stock; (1) the product comprising anti-A vaccine, (2) the product comprising anti-B vaccine; and (3) the product comprising anti-A and anti-B vaccine. In the case of vaccines against three diseases, seven different vaccines/combinations have to be made and stored. For four diseases, this number mounts to fifteen different vaccines/combinations. Consequently, it is often necessary to provide and maintain a large storage capacity.
U.S. Pat. Nos. 5,397,569 and 5,871,748 (Whitfill et al.) disclose a method for producing active immunity against Newcastle Disease virus (NDV) in avian subjects by administering, in-ovo, a vaccine complex comprised of a live vaccine virus and neutralizing antibodies bound thereto. Whitfill discloses that the ratio of virus to the neutralizing antibody or fragment thereof will determine the success of the immunization. However, for such a method to be applied effectively against NDV, a narrow range of values for that ratio must be maintained so that successful immunization will occur without killing the chicks. Moreover, the unpredictability in the titer using lyophilized NDV (as with other virus preparations) means that methods such as Whitfill's are not easily applicable for NDV. Therefore, a method which could ensure more exact titers of NDV and antibodies for preparing such a vaccine is desired.
Yet another disadvantage to prior vaccine formulations and their methods of production lie in the space-consuming nature of the actual freeze drying process. Typically, lyophilizing a vaccine first requires dispensing the solubilized vaccine into glass vials. The vials are then loosely capped with rubber stoppers and placed on trays in racks in a freeze drying chamber or condenser. It is difficult to concentrate very high doses of solubilized vaccine material in a very small volume.
As a result, the vials typically used in freeze drying for multiple vaccine doses always contain a relatively large volume of fluid. Efficient freeze drying requires that the fluid to be lyophilized exposes a large surface area to the vacuum. Therefore, since only the top of the frozen pellet is in contact with the vacuum, vaccines are freeze dried in relatively large glass bottles or vials, with a wide bottom. These vials are sometimes 5 centimeters high, wherein an additional 2 cm of height is needed for the rubber stoppers. Consequently, the ratio of product material to empty space in the freeze drying apparatus is extremely inefficient leading to a less cost effective production process.
Another disadvantage to conventional compositions for freeze dried vaccines formulated to be contained in glass vials is that the rubber stoppers can hinder free transfer of water molecules from the vial to the condenser during lyophilization. This can increase the partial pressure of sublimating gases inside the vial, thereby decreasing the efficiency of the lyophilization, as well as increase the risk of collapse of the lyophilized material. Glass also impedes heat transfer to further affect lyophilization efficiency. Formulating a vaccine that would eliminate both glass vials and stoppers by using trays in the lyophilization would increase efficiency. Moreover, freeze drying is very time-consuming. Vaccine components are typically kept just below the freezing point to decrease water sublimation time. However, a longer drying period leads almost inevitably to a decrease of titer. The glassvials with their stoppers and aluminum covers are generally seen as an encumbrance. In production terms they can represent more than 50 percent of the cost of the finished vaccine. In field situations, the diluent must be injected into the vial and the resultant solution extracted and diluted, if necessary for use. This is an inconvenience not always suited to on-site situations, such as in a chicken-shed. It is not uncommon for the operative in these situations to accidentally be selfinjected during this procedure. Yet another disadvantage to such vaccine packaging and preparation is that some of the concentrated solution will remain in the vial. Consequently, it is generally accepted that an overage in the contents is necessary to compensate for non-homogeneity and production losses, as well as losses incurred over storage periods. Glass vials must also be safely disposed of and can result in possible health and environmental hazards.
As a result, use of conventional freeze dried vaccine formulations involve complicated preparation techniques which are costly and difficult to implement in the field. Consequently, such conventional vaccine formulations are not well-suited for vaccinations in underdeveloped countries were economic and field conditions do not allow for costly campaigns, or in the case of mass immunizations which may be needed, such as in the defense of biological warfare, or other potential catastrophes or epidemics.
What is desired, therefore, is a vaccine formulation which obviates the use of glass vials for administration providing a less costly and bulky packaging alternative. What is also desired is a vaccine formulation and method of use which provides greater dosing accuracy and ease of use while maintaining stability, sterility, solubility and homogeneity for both single and multiple vaccine formulations. A vaccine formulation and improved immunizing method which facilitates more accurate, reproducible and efficient administration can provide the advantages of better field performance, increased safety, cost-effectiveness, less waste, and improved environmental compliance.